CA2091005C

CA2091005C - Microbiological process for hydroxylation of nitrogen-heterocyclic-carboxylic acids

Info

Publication number: CA2091005C
Application number: CA002091005A
Authority: CA
Inventors: Andreas Kiener; Andreas Tschech; Klaus Heinzmann; Andreas Tinschert
Original assignee: Lonza AG
Current assignee: Lonza AG
Priority date: 1992-03-04
Filing date: 1993-03-04
Publication date: 2001-09-25
Anticipated expiration: 2013-03-04
Also published as: ES2101887T3; LV10504B; LTIP377A; LT3114B; KR930019834A; DK0559116T3; CZ27293A3; CZ282939B6; CN1077748A; LV10504A; EP0559116A3; JPH06199797A; US5306630A; EP0559116B1; GR3024286T3; KR100274205B1; GEP19991710B; CA2091005A1; RU2122029C1; SK279322B6

Abstract

A microbiological process for the production of hydroxy-substituted nitrogen-containing heterocyclic carboxylic acids of the general formula:

(see formula I) and the soluble salts thereof, where R1 and R2 are the same or different and each represents a hydrogen atom, a halogen atom or a C1-C4 alkyl group, and X is a nitrogen atom or a CR3 group, where R3 is a hydrogen atom or a halogen atom.
The process uses the corresponding nitrogen-containing heterocyclic carboxylic acids as a starting material.
Microorganisms suitable for use in the process are also described.

Description

The present invention relates to a microbiological pracess for the production of hydroxy-substituted nitrogen-containing heterocyclic carboxylic acids of the general formula:
Rx x cooH
0 (z) and their soluble salts, where R~ and Rz each represents a hydrogen atom, a halogen atom, or a C~-C4 alkyl group; R1 and RZ can represent the same or different groups; and X
represents a nitrogen atom or a CR3 group where R3 represents a hydrogen atom or a halogen atom. The hydroxy-substituted nitrogen-containing heterocyclic carboxylic acids are produced from the corresponding nitrogen-containing heterocyclic carboxylic acids. The present invention also describes microorganisms suitable for use in the process.
Hereinafter reference to nitrogen-containing heterocyclic carboxylic acids includes the hydroxylated and non-hydroxylated acids, and soluble salts thereof, such as alkali or ammonium salts.

2-Hydroxy-substituted nitrogen-containing heterocyclic carboxylic acids are important intermediate products in active ingredient syntheses. For example, 2-hydroxynicotinic acid can be used as the starting material for the production of 2-chloronicotinic acid (U. S. Patent No. 4, 081, 451) which is an important intermediate in the production of pharmaceutical agents [Ann. Pharm. Fr., 38(3), (1980), pp. 243 to 252J.
Until now no microbiological process for the production of hydroxy-substituted nitrogen-containing heterocyclic carboxylic acids was known.

2~~~.(l~~

An object of the present invention is to provide a simple, ecological microbiological process for the production of 2-hydroxy-substituted nitrogen-containing heterocyclic carboxylic acids. These acids are difficult to produce by chemical processes.
According to the present invention there is provided a microbiological process for the production of a hydroxy-substituted nitrogen-containing heterocyclie carboxylic acid having the general formula:

(I) or a soluble salt thereof, where R~ and RZ are the same or different and each is a hydrogen atom, a halogen atom, or a C~-C4 alkyl group, and X is a nitrogen atom or a CR3 group where R3 is a hydrogen atom, or a halogen atom, which comprises converting a nitrogen-containing heterocyclic carboxylic acid of the general formula:

(II) R N

or a soluble salt thereof, wherein R~, RZ and X have the ' above-stated meanings, by means of a microorganism utilizing 6-methylnicotinic acid, containing a specific hydroxylase.
According to another aspect of the present invention, there is provided a microorganism capable of utilizing 6-methylnicotinic acid as the sole carbon, ~~~~.OOa nitrogen and energy source in the production of 2-hydroxy-6-methylnicotinic acid.
According to yet another aspect of the present invention, there is provided a hydroxy-substituted nitrogen-containing heterocyclic carboxylic acid of the general formula:
R X COOH

(I) where X is a nitrogen atom or a -CH- group, R~ arid RZ are the same or different and each is a hydrogen atom, a halogen atom, or a C~-C4 alkyl group, with the proviso that, when X is a nitrogen atom, R~ and RZ are not both hydrogen;
and when X is a -CH- group, R~ and RZ are both a chlorine atom.
Preferably such hydroxy-substituted nitrogen-containing heterocyclic carboxylic acid is 5,6-dichloro-2-hydroxynicotinic acid, 3-hydroxy-5-methylpyrazine carboxylic acid or 3-hydroxy-5-chloropyrazine carboxylic acid.
In the process of the present invention, a nitrogen-containing heterocyclic carboxylic acid of the general formula:

(II) R ~ N

where R~ and R2 are the same or different and each represents a hydrogen atom, a halogen atom, or a C~-C4 alkyl group; and X is a nitrogen atom or a CRS group, where R3 is a hydrogen atom or a halogen atom;
is converted by microorganisms utilizing 6-methylnicotinic acid, containing a specific hydroxylase, into a hydroxy nitrogen-heterocyclic-carboxylic acid of general formula:

(1) where R~, RZ and X have the above-mentioned meanings.
The microorganisms utilizing 6-methylnicotinic acid, containing a specific hydroxylase can be obtained, for example, by cultivating biomass using sewage sludge as an inoculum with 6-methylnicotinic acid. Only the microorganisms that can utilize 6-methylnicotinic acid as the sole carbon, nitrogen and energy source will proliferate.
Microorganisms that can specifically hydroxylate the sole carbon atom present between the functional acid group and the nitrogen atom are then isolated using techniques known to those skilled in the art. The isolated microorganisms having a specific hydroxylase, can completely catabolize 6-methylnicotinic acid to produce 2-hydroxy-6-methylnicotinic acid as an intermediate.
The microorganisms of the present invention can be isolated, for example, from sewage sludge or soil samples using the above-mentioned technique.
Preferably, microorganisms having the designation Ki101 (DSM 6920) as well as descendants and mutants thereof are used for the process. These microorganisms were deposited on February 10, 1992 with the Deutsche Sammlung fiir Mikroorganismen and Zellkulturen GmbH (German Collection for Microorganisms and Cell Cultures, Limited Liability Company), Mascheroderweg 1b, D-3300 Brunswick, Germany.
The scientific (taxonomic) description of Ki101 (DSM 6920) is as follows:
cell shape tiny rods width, micron 0.~-0.5 length, micron 1.0-1.5 mobility -flagella lo gram reaction -lysis by 3% KoH +
aminopeptidase (Cerny) +
spores oxidase +
catalase +
A genus could not be determined by 16S rRNA-sequence analysis.
The selection and cultivation of these microorganisms, with 6-methylnicotinic acid, utilizes methods known to those skilled in the art. Preferably the selection of the microorganisms is conducted under aerobic and static culture conditions.
The amount of 6-methylnicotinic acid used during the selection and the cultivation can be, for example, up to 1% by weight and preferably up to 0.5% by weight. The selection and cultivation steps are performed at a pH of 5 to 9, and at a temperature between 15° and 50°C.
Preferably the pH is in the range of 6 to 8 and the temperature is between 25° and 40°C.
The microorganisms are cultured in a mineral salt medium, preferably having a composition as described in Table 1.
When the optical density at 650 nm (ODbso) reaches 0.5 to 100, the microorganisms can be harvested according to methods known to those skilled in the art or, alternatively, the substrate, nitrogen-containing heterocyclic carboxylic acid, can be added directly to the microorganisms for the biotransformation reaction. The biotransformation is performed according to known techniques with nongrowing cells.
The substrate used has the general formula II and is, for example, nicotinic acid, pyrazine carboxylic acid or their halogenated or C~-CG alkylated derivatives. When the substrate is a halogenated nicotinic acid or a pyrazine carboxylic acid derivative, preferably the 6-chloronicotinic acid, 5,6-dichloronicotinic acid and 5-chloropyrazine carboxylic acid are hydroxylated. When the substrate is a Cy-C4 pyrazine carboxylic acid derivative, preferably the 5-methylpyrazine-carboxylic acid is hydroxylated.
The substrate for the biotransformation can be added continuously or all at once. Ideally, the substrate is added in such a way that the substrate concentration in the culture medium does not exceed 10 percent by weight, and preferably does not exceed 7 percent by weight.
The biotransformation, can be performed in a medium known to those skilled in the art. Preferably the biotransformation is performed in a low-molar phosphate buffer. Typically the biotransformation is performed with a microorganism suspension that has an optical density at 650 nm (ODbso) of 0.5 to 100, and preferably between 5 and , 50. The biotransformation is performed at a temperature of 15° to 50°C, and a pH of 5 to 9. Preferably the temperature is between 25° and 35°C and the pH is between 6.5 and 7.5.
After a reaction time of between 5 hours and 3 days, the hydroxylated product of general formu7.a I can then be isolated according to known methods, for example, by acidification of the cell-free supernatant.
As indicated above, the hydroxy-substituted nitrogen-containing heterocyclic carboxylic acids produced 2~~:~.~0~
according to the present invention have a general formula I:
Rl X COU~i N~ N OFi ( I ) where R~, Rz and X are as previously described.
Furthermore, the compounds of formula I in which X is a nitrogen atom, Ry and RZ are the same or different and each is a hydrogen atom, a halogen atom or a C~-C4 alkyl group, with the proviso that R~ and RZ do not simultaneously represent hydrogen are novel. As well, compounds (I) in which X is a CH group, R~ and Rz both represent a halogen atom are also novel. Preferred novel compounds are 5,6-dichloro-2-hydroxynicotinic acid, 3-hydroxy-5-methylpyrazine-carboxylic acid and 3-hydroxy-5-2o chloropyrazine carboxylic acid.
The following Examples illustrate the invention.
Example 1 Isolation of microorcranisms usinc~6-methyl-nicotinic acid 100 ml of the mineral salt medium of Table I
containing 1 mmol of 6-methylnicotinic acid was added to a 300 ml Erlenmayer flask and mixed with 10 ml of sewage sludge from the LONZA AG sewage treatment plant in Visp, Switzerland, or with soil samples from the LONZA-Werk, Visp, and incubated at 30°C under static conditions. The catabolism of 6-methylnicotinic acid was observed with UV-spectrum at 200 - 400 nm. After 10 days, 2-hydroxy-6-methylnicotinic acid was spectrophotometrically detectable in some batches. These cultures were then inoculated several times in fresh mineral salt medium. The cell suspensions forming 2-hydroxy-6-methylnicotini.c acid were then plated out on the same medium, containing 1.60 (w/v) ~~9~.~~~

of agar. The agar plates were incubated at 30°C in an atmosphere containing 4 % OZ and 96% N1. :Cndividual colonies were then transferred in liquid medium and incubated under static conditions. The bacteria strain with the designation Ki101 (DSM 6920) formed 2-hydroxy-6-methylnicotinic acid during growth in medium containing 6-methylnicotinic acid.
Example 2 Microbial oxidation of nicotinic acid to 2-hydroxynicotinic acid Microorganisms with the designation Ki101 (DSM
6920) were incubated in 800 ml of mineral salt medium (Table 1) with addition of 8 mmol of 6-methylnicotinic acid in a 1 1 Fernbach flask at 30 ° C on a shaker at 100 rpm.
The inoculum was 5% (v/v). After 5 days, the OD6so was approximately 0.5. The cells were harvested and washed once with 50 mM phosphate buffer, pH 7Ø The cells were then resuspended in 20 ml of 50 mM phosphate buffer, pH

7.0, containing 200 mg (1.38 mmol) of nicotinic acid-sodium salt. The OD65o of the suspension was 20. After 6 hours incubation on a shaker at 30°C, no more nicotinic acid could be detected by thin-layer chromatagraphy. The biomass was then centrifuged and the supernatant was acidified to pH 2.5 to precipitate the 2-hydroxynicotinic acid. No contamination in the isolated product could be detected by 'H-NMR (DMSO) analysis. 140 mg (1 mmol) of 2-hydroxynicotinic acid was obtained representing a yield of 72 percent relative to nicotinic acid.

~~~~.~lQ

Table Mineral salt medium stock solutions Stock solution 1:

NaH2P04 2H20 156 . g NH4C1 10.U g KZSO4 1. 2 g pH to be adjusted with KOH to 7.0 g in distilled water 500.0 ml Stock solution 2:

p-aminobenzoic acid 8.0 mg D-biotin 2.0 mg nicotinic acid 20.0 mg Ca-D-pantothenate 10.0 mg pyridoxalhydrochloride 30.0 mg thiamindichloride 20.0 mg cyanocobalamin 10,0 ma in distilled water 100.0 ml, sterilized by Filtration Stock solution 3:
HCl (37%) 7.0 ml FeClz 4H20 1. 5 g znCl2 0.07 g MnCl2 4Hz0 0 .1 g H3B03 0.006 g CoCl2 6H20 0 .19 g CuClz 7H20 0 . 002 g NiCl2 6H2O 0 . 024 g Na2MoO4 2H20 0 . 03 6 a in distilled water 1000.0 ml, sterilized in an autaclave at 121C

for 20 minutes.

Stock solution 4:
NaOH 0.5 g NaZSe03 ~ 5HZ0 0 . 003 g Na2W04~2Hz0 0.004 a in distilled water 1000.0 ml; sterilized in an autoclave at 121°C
for 20 minutes.
Stock solution 5:
MgC126HZ0 40.0 g CaCl2 ~ 2H20 5 . 0 a in distilled water 200.0 ml, sterilized in an autoclave at 121°C
for 20 minutes.
6-Methvlnicotinic acid (500 mM):
6-methylnicotinic acid-sodium salt 80.0 a in distilled water 1000.0 ml, sterilized in an autoclave at 121°C
for 20 minutes.
Growth medium 10 mM of 6-methylnicotinic acid:
solution 1 25.0 ml 6-methylnicotinic acid 20.0 ml , in distilled water 1000.0 ml, sterilized in an autoclave at 121°C
for 20 minutes.
After the sterilization:
solution 2 0.5 ml solution 3 1.0 ml solution 4 1.0 ml solution 5 0.5 ml were added.

2~~~~~

Examples 3 to 7 Examples 3 to 7 were performed according to the procedure of Example 2. The results are set out in Tables 2 and 3.
Table 2 Example Starting material centration OD65oIncubation Con Time (hours) 3 6-chloronicotinic- 1% (w/v) 20 16 acid sodium salt 4 5,6-dichloronicotinic1% (w/v) 20 16 acid sodium salt 5 pyrazine-carboxylic 0.3% (w/v) 5 24 acid sodium salt 6 5-methyl-pyrazine 0.3% (w/v) 5 24 carboxylic acid sodium salt 7 5-chloro-pyrazine 0.3% (w/v) 5 24 carboxylic acid sodium salt 20~~~~~

Table 3 Example Product Yield 3 6-chloro-2-hydroxynicotinic acid700 (isolated) 4 5,6-dichloro-2-hydroxynicotinic 20% (analytic) acid 5 3-hydroxypyrazine carboxylic 70% (analytic) acid 6 3-hydroxy-5-methyl-pyrazine- 80% (analytic) carboxylic acid 7 3-hydroxy-5-chloro-pyrazine- 50% (analytic) carboxylic acid Analytical data for Example 6:
(3-Hydroxy-5-methyl-pyrazine carboxylic acid) ~H-NMR: (DMSO, 400 MHz) 8 in ppm 2.4, s; 2.6; s; 3.8, s; 7.8, s.
~3C-NMR: (DMSO, 100.5 MHz) d in ppm 20, t; 40, m; 130, s; 134, s; 155, s; 164, s; 170, s.
Ana ~tical data for Example 7:
(3-Hydroxy-5-chloro-pyrazine carboxylic acid) 'H-NMR: (DZO and dioxane, 400 MHz) d in ppm 3.8, s; 4.7, s; 8.0, s.
~3C-NMR: (D20 and dioxane, 100.5 MHz) 8 in ppm 132, s; 133, s; 145, s; 149, s; 164, s; 172, s.

Claims

1. A microbiological process for the production of a hydroxy-substituted nitrogen-containing heterocyclic carboxylic acid having the general formula:

or a soluble salt thereof, where R1 and R2 are the same or different and each is a hydrogen atom, a halogen atom, or a C1-C4 alkyl group, and X is a nitrogen atom or a CR3 group where R3 is a hydrogen atom, or a halogen atom, which comprises converting a nitrogen-containing heterocyclic carboxylic acid of the general formula:

or a soluble salt thereof, wherein R1, R2 and X have the above-stated meanings, into a product according to formula I by microorganism DSM6920, or a mutant or descendant thereof, which is capable of effecting said conversion, said microorganism DSM6920, or mutant or descendant thereof, being capable of utilizing 6-methylnicotinic acid and containing a hydroxylase which is specific for the conversion of a compound according to formula II, into a compound of formula I.

2. The process according to claim 1, wherein a nitrogen-containing heterocyclic carboxylic acid of the general formula II is used as a substrate, wherein R1 and R2 are the same or different and each is a hydrogen atom, a chlorine. atom, or a methyl group and X is a nitrogen atom or a -CH- group.

3. The process according to claim 1 or 2, wherein the reaction takes place by a one-time or continuous substrate addition so that the substrate concentration in the culture medium does not exceed about 10 percent by weight.

4. The process according to any one of claims 1 to 3, wherein the reaction is performed at a temperature of 15° to 50°C and at a pH of 5 to 9.

5. A microorganism DSM 6920, or a mutant or descendant thereof, which is capable of utilizing 6-methylnicotinic acid as the sole carbon, nitrogen and energy source in the production of 2-hydroxy-6-methylnicotinic acid.

6. A hydroxy-substituted nitrogen-containing heterocyclic carboxylic acid of the general formula:

where X is a nitrogen atom or a -CH- group, R1 and R2 are the same or different and each is a hydrogen atom, a halogen atom, or a C1-C4 alkyl group, with the proviso that, when X is a nitrogen atom, R1 and R2 are not both hydrogen; and when X is a -CH- group, R1 and R2 are both a chlorine atom.

7: 5,6-Dichloro-2-hydroxynicotinic acid.

8. 3-Hydroxy-5-methylpyrazine carboxylic acid.

9. 3-Hydroxy-5-chloropyrazine carboxylic acid.